This invention relates to three novel monoclonal antibodies (mAbs) termed SN8, SN8a and SN8b which are directed toward individually different epitopes of the same antigen molecule, which is primarily expressed on B-cell type B prolymphocytic leukemia (PLL) cells, B non-Hodgkins' lymphoma (NHL) cells, B acute lymphoblastic leukemia (ALL), and to methods of using the monoclonal antibodies, in whole or in part, for the diagnosis and therapy of various human leukemia-lymphomas (HLL), especially PLL and NHL.
Prolymphocytic leukemia (PLL) was initially reported in 1974 by Galton et al. as a variant of chronic lymphocytic leukemia (CLL), but with distinct clinical and laboratory features ("Prolymphocytic Leukemia", British J. Haematol, 27: 7, 1974). The distinction between PLL and CLL is clinically important because PLL is a relentlessly progressive disease with a worse prognosis and usually resistant to the chemotherapy that is often effective in CLL (Catovsky, "Prolymphocytic and hairy cell leukemias", in Henderson E. S., Lister T. A. (eds): Leukemia, Philadelphia, Pa., Saunders, 1990, p. 639). The majority (approximately 75%) of PLL cases are of B cell origin as are most cases (approximately 98%) of CLL (Foon et al., "Immunologic classification of leukemia and lymphoma", Blood, 68: 1, 1986). Furthermore, B PLL appears to be closely related to hairy cell leukemia (HCL) in the differentiation pathway of B cell ontogeny.
SN8 was capable of effectively distinguishing B PLL from B CLL as well as from hairy cell leukemia (HCL) cell specimens. SN8a and SN8b also showed a selective reactivity with B PLL and B NHL samples. However, these mAbs reacted with higher percentages of CLL samples compared to SN8. Thus, the degree of PLL selectivity of these mAbs is less than that of SN8.
Recent study of the chemical structure of the SN8 antigen revealed that SN8 antigen is a novel heterodimer antigen which constitutes the human B cell antigen receptor complex together with cell membrane immunoglobulin.
Antigen receptors on B lymphocytes (B cells) play a central role in the immune regulation in animals and humans. In the case of the murine B cells, two disulfide-linked transmembrane molecules, encoded by the mb-1 and B29 genes, have been recently defined as integral components of the antigen receptors on murine B cells. The mb-1 and B29 products are termed Ig-.alpha. and Ig-.beta., respectively. A variant form of the Ig-.beta. was called Ig-.gamma.. The disulfide-linked heterodimer .alpha.-.beta. molecules on B cells are noncovalently associated with cell membrane immunoglobulins (mIgs). Recent studies by several investigators suggest that a similar situation exists for the antigen receptor complex on human B cells. However, the human homologues of murine mb-1 and B29 gene products have not been definitely identified.
The present invention discloses the generation and characterization of three mAbs termed SN8, SN8a and SN8b that were generated by immunizing two mice with an isolated PLL antigen preparation. These mAbs, particularly SN8, showed a highly selective reactivity with B PLL and B NHL. The surface antigen defined by the SN8 series mAbs was identified as a covalently-linked heterodimer (gp49/40). Comparison of SN8 series mAbs and SN8 antigen with the reported mAbs and their antigens suggests that SN8 series mAbs define a novel B-cell antigen.
This conclusion was corroborated by the determination of the amino-terminal amino acid sequences of the individual components (.alpha. and .beta. chains) of the SN8 antigen. The amino acid sequence study revealed unequivocally that the .alpha. (47-49 kD) and .beta. chain (37-40 kD) components of the SN8 antigen are the human mb-1 and B29 proteins, respectively. In the amino acid sequence study, the non-radiolabeled SN8 antigen was isolated from human B leukemia cells using SN8 immunoaffinity column. Use of SN8 was indispensible for the isolation and immunological analysis of the human mb-1 and B29 heterodimer in the present study. For unknown reasons, it is extremely difficult to generate a mAb defining the human B29 or mb-1 protein by conventional approach, i.e., immunizing mice with human B cells. Thus, few mAbs specific for an extracellular epitope of the human B29 or mb-1 protein have been previously reported. An exception is a recent report by Nakamura et al. ("Heterogeneity of immunoglobulin-associated molecules on human B cells identified by monoclonal antibodies", Proc. Natl. Acad. Sci. USA, 89: 8522, 1992) who generated two mAbs defining the putative human equivalent of the mouse B29 protein. They used an elaborate procedure including repeated injections of mice with proteins separated by SDS-PAGE. Nakamura et al. used the phrase "putative human equivalent of the mouse B29 protein" because they had neither characterized the antigen chemically nor determined the antigen's amino acid sequence. Also, it appears that no amino acid sequence of the human B29 or mb-1 proteins has been reported prior to the present invention.
Since the present mAbs show a highly selective reactivity as well as high binding avidity (SN8 and SN8b) and are of IgG1 isotype, they may be useful for the diagnosis of B leukemia/lymphoma, for studying the pathogenesis of PLL and perhaps for studying normal human B cell differentiation.
In addition, ricin A-chain (RA) conjugates of the three mAbs are all effective for the specific killing of SN8 antigen-expressing cells. Furthermore, binding of these mAbs to the cell surface antigen induced no significant (SN8 and SN8b) or only a small (SN8a) down-regulation of antigen expression. The results suggest the potential of these mAbs as a specific delivery vehicle of cytotoxic agents to the SN8 antigen-expressing target cells.
The B cell antigen receptor complex plays a central role in the immune regulation in the human. Therefore, SN8 series mAbs will have good potential for the diagnosis and therapy of human diseases involving immunological functions as well as for the immunological manipulation of human B cells, e.g., inducing immune unresponsiveness.
BRIEF DESCRIPTION OF THE INVENTION
The invention comprises leukemia lymphoma reactive monoclonal antibodies directed toward individually different epitopes of the same antigen molecule which, relative to normal peripheral blood cells, strongly reacts with one or more leukemia lymphoma cell specimens from the group consisting of B prolymphocytic leukemia cells, B non-Hodgkin's lymphoma cells, B chronic lymphocytic leukemia cells, B hairy cell leukemia cells, and B acute lymphoblastic leukemia cells.
More particularly, three new IgG1-.kappa. monoclonal antibodies (mAbs), termed SN8, SN8a and SN8b, were generated by the use of an unconventional approach, i.e., utilizing an isolated B PLL antigen preparation to immunize mice. These mAbs, particularly SN8, showed a highly selective reactivity to B PLL and B non-Hodgkin's lymphoma among various human leukemia-lymphoma specimens tested, e.g., SN8 was capable of effectively distinguishing B PLL from B CLL as well as from HCL cell specimens. SN8 may be produced by hybridoma cell line 3A2-2E7 and clones thereof, SN8a may be produced by hybridoma cell line 3B3-1D2 and clones thereof and SN8b may be produced by hybridoma cell line Q6-1D5 and clones thereof. It is understood that the monoclonal antibodies of the invention are intended to include reactive fragments thereof, including F(ab').sub.2, Fab' Fab Fv, Fd' and Fd.
The invention further includes a diagnostic kit comprising monoclonal antibodies of the invention within a package.
The invention further comprises monoclonal antibodies which are directly or indirectly attached or complexed with a compound having a site suitable for attachment or complexing therewith which compound is selected from the group consisting of drugs, toxins or fragments thereof, growth suppressing biological response modifiers, enzymes, liposomes, radioactive agents and antibodies.
The invention also includes the methods for using the monoclonal antibodies for detecting and treating leukemia/lymphoma disease, as well as methods for preparation of the monoclonal antibodies and kits containing the same are also within the scope of the invention.
Finally, the invention also relates to the isolation and chemical identification of the human homologues of the murine mb-1 and B29 proteins of B cell antigen receptor complex. The amino-terminal amino acid sequences of the mb-1 and B29 gene products were determined using the antigen which was isolated using the monoclonal antibody SN8.